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Results for “Somatic Hypermutation, Immunoglobulin”

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PubMed2005

The translesion DNA polymerase theta plays a dominant role in immunoglobulin gene somatic hypermutation.

Immunoglobulin (Ig) somatic hypermutation (SHM) critically underlies the generation of high-affinity antibodies. Mutations can be introduced by error-prone polymerases such as polymerase zeta (Rev3), a mispair extender, and polymerase eta, a mispair inserter with a preference for dA/dT, while repairing DNA lesions initiated by AID-mediated deamination of dC to yield dU:dG mismatches. The partial impairment of SHM observed in the absence of these polymerases led us to hypothesize a main role for another translesion DNA polymerase. Here, we show that deletion in C57BL/6J mice of the translesion polymerase theta, which possesses a dual nucleotide mispair inserter-extender function, results in greater than 60% decrease of mutations in antigen-selected V186.2DJ(H) transcripts and greater than 80% decrease in mutations in the Ig H chain intronic J(H)4-iEmu sequence, together with significant alterations in the spectrum of the residual mutations. Thus, polymerase theta plays a dominant role in SHM, possibly by introducing mismatches while bypassing abasic sites generated by UDG-mediated deglycosylation of AID-effected dU, by extending DNA past such abasic sites and by synthesizing DNA during dU:dG mismatch repair.

Animals↗

PubMed2002

Somatic immunoglobulin hypermutation.

Immunoglobulin hypermutation provides the structural correlate for the affinity maturation of the antibody response. Characteristic modalities of this mechanism include a preponderance of point-mutations with prevalence of transitions over transversions, and the mutational hotspot RGYW sequence. Recent evidence suggests a mechanism whereby DNA-breaks induce error-prone DNA synthesis in immunoglobulin V(D)J regions by error-prone DNA polymerases. The nature of the targeting mechanism and the trans-factors effecting such breaks and their repair remain to be determined.

Animals↗

PubMed2003

Immunoglobulin somatic hypermutation: double-strand DNA breaks, AID and error-prone DNA repair.

Somatic hypermutation (SHM) is critical for antibody affinity maturation and the generation of memory B cells. Somatic mutations consist mainly of single nucleotide changes with rare insertions and deletions. Such changes would be introduced during error-prone repair of lesions involving single-strand DNA breaks (SSBs) or, more likely, double-strand DNA breaks (DSBs), as DSBs occur exclusively in genes that have the potentials to undergo SHM. In the human, such genes include Ig V, BCL6, and c-MYC. In these germline genes, DSBs are blunt. In rearranged Ig V, BCL6, and translocated c-MYC genes, blunt DSBs are processed to yield resected DNA ends. This process is dependent on the expression of activation-induced cytidine deaminase (AID), which is selectively expressed upon CD40-signaling in hypermutating B cells. CD40-induced and AID-dependent free 5'- and 3'-staggered DNA ends critically channel the repair of DSBs through the homologous recombination (HR) repair pathway. During HR, the modulation of critical translesion DNA polymerases, as signaled by cross-linking of the B cell receptor (BCR) for antigen, leads to the insertions of mismatches, i.e., mutations. The nature of DSBs, the possible roles of AID in the modification of DSBs and that of the translesion DNA polymerases zeta and iota in the subsequent repair process that lead to the insertions of mutations are discussed here within the context of an integrated model of SHM.

Animals↗

PubMed2005

DNA cleavage in immunoglobulin somatic hypermutation depends on de novo protein synthesis but not on uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requires de novo protein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.

Animals↗

PubMed2005

Intricate targeting of immunoglobulin somatic hypermutation maximizes the efficiency of affinity maturation.

It is believed that immunoglobulin-variable region gene (IgV) somatic hypermutation (SHM) is initiated by activation-induced cytidine deaminase (AID) upon deamination of cytidine to deoxyuracil. Patch-excision repair of these lesions involving error prone DNA polymerases such as poleta causes mutations at all base positions. If not repaired, the deaminated nucleotides on the coding and noncoding strands result in C-to-T and G-to-A exchanges, respectively. Herein it is reported that IgV gene evolution has been considerably influenced by the need to accommodate extensive C deaminations and the resulting accumulation of C-to-T and G-to-A exchanges. Although seemingly counterintuitive, the precise placement of C and G nucleotides causes most C-to-T and G-to-A mutations to be silent or conservative. We hypothesize that without intricate positioning of C and G nucleotides the efficiency of affinity maturation would be significantly reduced due to a dominance of replacements caused by C and G transition mutations. The complexity of these evolved biases in codon use are compounded by the precise concomitant hotspot/coldspot targeting of AID activity and Poleta errors to maximize SHM in the CDRs and minimize mutations in the FWRs.

B-Lymphocytes↗

PubMed2003

By-products of immunoglobulin somatic hypermutation.

The antigen receptor loci are the only loci in humans to undergo programmed somatic gene modification. Although aberrant V(D)J integration and class switch recombination can both give rise to chromosomal translocations, a role for somatic hypermutation in such genomic rearrangements has been suggested but is less clearly established. To characterize the types of by-products of somatic hypermutation, we analyzed aberrant rearrangements involving the immunoglobulin loci in a human B-cell line (Ramos) that performs Ig V gene hypermutation constitutively during culture. Single-nucleotide substitutions account for 95% of the mutational events in the VH gene, with small deletions and duplications accounting for most of the remaining mutations. However, larger genetic alterations can be detected at low frequency, accounting for 0.5% of VH-inactivating mutations. Examples include a large (13 kb) deletion, which entirely removes the expressed VH gene; a 3-kb deletion extending from the functional VHDJH into an upstream VH (generating a hybrid VHDJH gene reminiscent of VH replacement); and an ectopic insertion into the VH from chromosome 1. The results support the proposal that aberrant antibody hypermutation can lead to gross genomic alterations but indicate that such events are rare.

Base Sequence↗

PubMed2002

Mice deficient for the type II topoisomerase-like DNA transesterase Spo11 show normal immunoglobulin somatic hypermutation and class switching.

Somatic hypermutation in B cells undergoing T cell dependent immune responses generates high affinity antibodies that provide protective immunity. B cells also switch from the expression of immunoglobulin (Ig) M and IgD to that of other Ig classes through somatic DNA recombination. Recent work has implicated DNA strand breaks, possibly DNA double strand breaks (DSB), as the initiating lesions in both class switch recombination and hypermutation, although the etiology of these lesions is not understood. Spo11, a protein structurally related to archaeal type II topoisomerases, generates DSB that initiate meiotic recombination. This characteristic, together with its expression pattern, marks this enzyme as a potential candidate for the initiation of hypermutation, and perhaps also for Ig class switching. To investigate whether Spo11 is involved in these processes, we studied the T cell dependent immune response of Spo11-deficient (Spo11(-/-)) mice against the hapten nitrophenyl (NP). We found that V186.2-bearing IgG1 transcripts had normal levels and patterns of somatic hypermutation. Furthermore, Spo11(-/-) mice showed normal serum levels of all Ig isotypes. These results indicate that Spo11 is not required for Ig hypermutation or class switch recombination.

Animals↗

PubMed2005

DNA lesions and repair in immunoglobulin class switch recombination and somatic hypermutation.

Immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are critical for the maturation of the antibody response. These processes endow antibodies with increased antigen-binding affinity and acquisition of new biological effector functions, thereby underlying the generation of memory B cells and plasma cells. They are dependent on the generation of specific DNA lesions and the intervention of activation-induced cytidine deaminase as well as newly identified translesion DNA polymerases, which are expressed in germinal center B cells. DNA lesions include mismatches, abasic sites, nicks, single-strand breaks, and double-strand breaks (DSBs). DSBs in the switch (S) region DNA are critical for CSR, but they also occur in V(D)J regions and possibly contribute to the events that lead to SHM. The nature of the DSBs in the Ig locus, their generation, and the repair processes that they trigger and that are responsible for their regulation remain poorly understood. Aberrant regulation of these events can result in chromosomal breaks and translocations, which are significant steps in B-cell neoplastic transformation.

Animals↗

PubMed2005

DNA polymerases and somatic hypermutation of immunoglobulin genes.

Somatic hypermutation of immunoglobulin variable genes, which increases antibody diversity, is initiated by the activation-induced cytosine deaminase (AID) protein. The current DNA-deamination model posits that AID deaminates cytosine to uracil in DNA, and that mutations are generated by DNA polymerases during replication or repair of the uracil residue. Mutations could arise as follows: by DNA replicating past the uracil; by removing the uracil with a uracil glycosylase and replicating past the resulting abasic site with a low-fidelity polymerase; or by repairing the uracil and synthesizing a DNA-repair patch downstream using a low-fidelity polymerase. In this review, we summarize the biochemical properties of specialized DNA polymerases in mammalian cells and discuss their participation in the mechanisms of hypermutation. Many recent studies have examined mice deficient in the genes that encode various DNA polymerases, and have shown that DNA polymerase H (POLH) contributes to hypermutation, whereas POLI, POLK and several other enzymes do not have major roles. The low-fidelity enzyme POLQ has been proposed as another candidate polymerase because it can efficiently bypass abasic sites and recent evidence indicates that it might participate in hypermutation.

Animals↗

PubMed1996

The molecular basis of somatic hypermutation of immunoglobulin genes.

Somatic hypermutation amplifies the variable region repertoire of immunoglobulin genes. Recent experimental evidence has thrown light on various molecular models of somatic hypermutation. A link between somatic hypermutation and transcription coupled DNA repair is shaping up.

Animals↗

PubMed2001

Somatic hypermutation of immunoglobulin and non-immunoglobulin genes.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a highly specific mechanism restricted to B lymphocytes during only a few cell generations. Data presented here suggest that transcription of the target genes is required, but not sufficient for SHM. Presumably, cis-acting elements, such as those present in the Ig enhancers, are required to target a mutator factor (MuF) to Ig and human BCL-6 genes. It is postulated that the MuF travels with the transcribing RNA polymerase and is deposited on the target gene when the polymerase pauses. Point mutations, and rare deletions and insertions, are created by the combined actions of MuF and certain DNA polymerases. A subset of the mutations is corrected during SHM by DNA mismatch repair.

Animals↗

PubMed2004

On the molecular mechanism of somatic hypermutation of rearranged immunoglobulin genes.

Somatic hypermutation (SHM) diversifies the genes that encode immunoglobulin variable regions in antigen-activated germinal centre B lymphocytes. Available evidence strongly suggests that DNA deamination potentiates phase I SHM and subsequently triggers phase II SHM. A concise review of this evidence is followed by a detailed critique of two possible models which suggest that polymerase-eta potentiates phase II SHM via either its DNA-dependent or its RNA-dependent DNA synthetic activity. Quantitative analysis, in the context of extant data that define the features of SHM, favours the RNA-dependent mechanism.

Animals↗

PubMed1996

Somatic hypermutation of immunoglobulin genes.

The relationship between somatic hypermutation and affinity maturation in the mouse is delineated. Recent work on the anatomical and cellular site of this process is surveyed. The molecular characteristics of somatic hypermutation are described in terms of the region mutated and the distinctive patterns of nucleotide changes that are observed. The results of experiments utilizing transgenic mice to find out the minimum cis-acting sequences required to recruit hypermutation are summarized. The hypothesis that V gene sequences have evolved in order to target mutation to certain sites but not others is discussed. The use that different species make of somatic hypermutation to generate either the primary or secondary B cell repertoire is considered. Possible molecular mechanisms for the hypermutation process and future goals of research are outlined.

Animals↗

PubMed2002

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota.

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

Antibody Affinity↗

PubMed2002

Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity.

Somatic hypermutation is critical for the generation of high-affinity antibodies and effective immune responses, but its molecular mechanism remains poorly understood. Recent studies have identified DNA strand lesions associated with the hypermutation process and suggested the involvement of specific repair molecules and pathways. Particularly exciting has been the discovery of a putative RNA editing enzyme, the activation-induced cytidine deaminase (AID), that is required for all immunoglobulin gene-specific modification reactions (somatic hypermutation, class switch recombination, and gene conversion). Parallels between these three reactions are considered in light of recent advances.

Animals↗

PubMed2004

HBL-3 cell line, derived from precursor B-cell lymphoblastic leukemia, lacks somatic hypermutation of immunoglobulin heavy chain variable region genes.

Somatic mutation (SM) analysis provides a useful tool for understanding the stages at which neoplastic differentiate from normal B-cells. B-cell precursor neoplasms are considered to be somatically premutational. However, the variable frequency of SM of the variable region (VH) genes has been described in cases of precursor B-cell acute lymphoblastic leukemia (PB-ALL). To better characterize PB-ALL based on the differentiation stage, we investigated the SM of the VH genes expressed by tumor cells of the surface immunoglobulin (sIg)(-) HBL-3 cell line derived from childhood PB-ALL. In the HBL-3 cell line, the rearranged Ig heavy chain VH gene sequence showed no SM in the complementarity-determining regions of 1, 2, and 3, or in the framework regions of 1, 2, and 3 relative to the putative germline VH gene sequences. In addition, the VH segment of HBL-3 cells showed no intraclonal sequence heterogeneity, indicating ongoing SM. Our data demonstrated that HBL-3 cells express unmutated and developmentally regulated rearrangement of VH genes at the stage of B-cell precursor cells. HBL-3 cells, which are derived only from the sIg(-) PB-ALL, showed that SM cannot be recognized in VH genes of tumor cells before the expression of sIg.

Base Sequence↗

PubMed2006

Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences.

It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light chain (IgL) transcriptional control elements in GCV/SHM in the chicken B cell line DT40. Promoter substitution experiments led to identification of a strong RNA polymerase II promoter incapable of supporting efficient GCV/SHM. This surprising finding indicates that high levels of transcription are not sufficient for robust GCV/SHM in Ig loci. Deletion of the IgL enhancer in a context in which high-level transcription was not compromised showed that the enhancer is not necessary for GCV/SHM. Our results indicate that cis-acting elements are important for Ig gene diversification, and we propose that targeting specificity is achieved through the combined action of several Ig locus elements that include the promoter.

Actins↗

PubMed2001

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation.

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.

Animals↗